The affinity labeling of delta 5-3-Ketosteroid isomerase from Pseudomonas testosteroni by photoexcited delta4-3-Ketosteroids is being investigated with regard to mechanism and products. Wavelength dependency studies have provided evidence that inactivation proceeds via photon absorption by bound steroid rather than by unbound steroid or a protein chromophore. Changes in the electrophoretic mobility and the amino acid composition of the enzyme and a peptide fragment derived therefrom have implicated either aspartic acid residues 32, 33 or 38 as the site of photochemical modification responsible for enzyme inactivation. Preliminary evidence suggests that one of these residues becomes converted to alanine during photinactivation. Delta 5-3-Ketosteroid isomerase has been purified to homogeneity from Pseudomonas putida. This enzyme is distinctly different from the testosteroni enzyme in amino acid composition and in a requirement for maintenance of thiol groups in a reduced state. Structural studies of this enzyme are to be initiated and attempts to photoaffinity label its steroid binding site (as with the testosteroni enzyme) are planned. Similiar attempts will be made with steroid binding proteins of animal and human sera. A modification of the technique of peptide mapping which adapts it to preparative purposes has been developed and will be further refined. The technique avoids exposure of a major portion of the peptides on the map to chemical reagents used for peptide visualization. BIBLIOGRAPHIC REFERENCES: Martyr, R.J. and Benisek, W.F. (1975) "Chemical Modification of Amino Acid Residues Associated with the Delta 4-3-Ketosteroid-Dependent Photoactivation of delta 5-3-Ketosteroid Isomerase. J. Biol. Chem. 250, 1218-1222. Lee, Y.M. and Benisek, W.F. (1976) "Inactivation of Phosphorylase b by Potassium Ferrate, A New Reactive Analogue of the Phosphate Group. J. Biol. Chem., in press.